摘要:
提取赤点石斑鱼(Epinephelus akaara)基因组DNA,经BamH I和Hind Ⅲ双酶切并电泳回收300~1 000 bp的片段与经BamH I和Hind Ⅲ双酶切的pUC18质粒重组,转化感受态大肠杆菌JM109,以α互补法筛选重组阳性克隆,建立赤点石斑鱼部分基因组文库。应用PCR混合池法筛选含核心序列为(CA)n和(GA)n微卫星位点的阳性克隆,经DNA测序,得到61个含微卫星序列的克隆,占总克隆数的2.89%。61个用于测序的克隆中共有111个不同类型的微卫星序列,其中(CA/TG)n重复类型57个,占51.4%;(AG/TC)n重复类型35个,占31.5%;其他重复类型19个,占17.1%。完美型、非完美型及复合型序列分别占48.7%、41.4%和9.9%。并在GenBank上注册了22个微卫星序列。
Abstract:
A partial genomic library of Epinephelus akaara was constructed and a PCR-based strategy was developed to screen the microsatellites in the library.A total of 2112 recombinant colonies were picked and regrown overnight in 96-well cell culture plates with 100 μL of LB medium plus ampicillin each well at 37℃.Twenty two plates were obtained and there were 20 mixing-pools in each plate.A PCR-based screening library method was used to screen these mixing-pools via the universal primer of M13 and repeat motif primers(CA)7C and(GA)7G,respectively.There were 111 microsatellites isolated after 61 positive clones were sequenced.The results indicated that microsatellite sequences characterized by(AC/GT)n and(GA/CT)n were abundant in genomic DNA of Epinephelus akaara.A portion of microsatellites repeat motifs and their flanking sequences were described.Among the 111 microsatellites,there were 54 perfect ones(48.7%),46 imperfect ones(41.4%),and 11 compound ones(9.9%).The 22 microsatellites sequences were submitted to the GenBank.It is supposed that these loci could be used for further genetic diversity and population structure analysis of Epinephelus akaara.
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